CN107449900B - Multi-residue detection antigen and antibody of carbamate and pyrethroid, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a preparation method and application of a carbamate and pyrethroid multi-residue detection antigen and antibody. The invention fills the long-standing technical gap in the field, and provides a combined antigen for simultaneously detecting the multi-residues of carbamates and pyrethroids. Methods for the detection of multiresidues of esters. with broadly application foreground. Said carbamate and pyrethroid multi-residue detection antigen, its structural formula is shown in formula (I):

(I).

Description

Antigen, antibody and preparation method thereof for multi-residue detection of carbamate and pyrethroid application

technical field

The invention relates to the technical field of food safety, and more particularly, to a carbamate and pyrethroid multi-residue detection antigen, an antibody, and a preparation method and application thereof.

Background technique

my country is a big agricultural country, and the output and consumption of agricultural products are in the forefront of the world every year. However, the problem of pesticide residues has always plagued my country's agricultural development. Carbamate pesticides and pyrethroid pesticides are two common types of pesticides in agricultural production in my country. For this reason, researchers have developed a series of detection methods for these two types of pesticides. Among them, immunodetection methods have high sensitivity and specificity. , simple pretreatment, short inspection period, suitable for large-scale sample detection and other advantages, has become a research hotspot in recent years. However, limited by the specific mindset, the existing immunoassay methods are only suitable for the detection of one type of pesticides, and cannot meet the detection of multiple pesticides at the same time, resulting in useless duplication of labor and waste of resources. Therefore, it is of great significance to develop a rapid detection method that can simultaneously detect carbamate pesticides and pyrethroid pesticides.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the existing technical blank of simultaneous detection and analysis of carbamate pesticides and pyrethroid pesticide residues, and to provide an antigen for simultaneous detection of carbamate pesticides and pyrethroid pesticide residues.

Another technical problem to be solved by the present invention is the preparation method of hapten and antigen for simultaneously detecting the residues of carbamate pesticides and pyrethroid pesticides.

Another technical problem to be solved by the present invention is to provide an antibody for simultaneously detecting the residues of carbamate pesticides and pyrethroid pesticides.

Another technical problem to be solved by the present invention is the application of antigens and antibodies for simultaneous detection of carbamate pesticides and pyrethroid pesticide residues.

The object of the present invention is achieved through the following technical solutions:

Provided is a combination antigen of carbamate and pyrethroid, whose structural formula is shown in formula (I):

Preferably, the carrier protein is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

The present invention also provides a method for preparing the carbamate and pyrethroid multi-residue antigens. The carbamate hapten is coupled with a carrier protein by an active ester method, and then the coupled product continues to pass through the active ester. The method is coupled with pyrethroid hapten to prepare carbamate and pyrethroid multi-residue detection antigens.

Specifically, the preparation method of described carbamates and pyrethroids combined antigen, comprises the following steps:

S1. Dissolve the carbamate hapten in DMF to form a mixed solution, then add EDC and NHS, and stir the reaction at room temperature; after the reaction is completed, add the above reaction solution to the phosphate of the carrier protein (pH7.4) In the buffer solution, continue the reaction, and then dialyze the reaction solution at room temperature to obtain the carbamate complete antigen;

S2. Dissolve the pyrethroid hapten in DMF to form a mixed solution, then add EDC and NHS, and stir the reaction at room temperature;

S3. Dissolve the carbamate complete antigen prepared in step S1 in the DMF solution, then add the reaction solution prepared in step S2, react at room temperature, and after the reaction is completed, the reaction solution will be dialyzed to obtain the described Antigens for multi-residue detection of carbamates and pyrethroids.

Preferably, the mass ratio of the carbamate hapten described in step S1 to the carrier protein is 12:1.

Preferably, the reaction time of the carbamate hapten in step S1 with EDC and NHS is 8-24 hours.

Preferably, the reaction time between the carbamate hapten and the carrier protein in step S1 is 6-24 hours.

Preferably, the reaction time in step S2 is 8-12 h.

Preferably, the mass ratio of the pyrethroid hapten to the carbamate antigen described in step S3 is 10:1.

Preferably, the reaction time in step S3 is 6-8h.

In the above preparation method, the mass ratio of DMF, EDC and NHS is determined according to 10:2:1.

In the above preparation method, the composition of the phosphate buffer solution of the carrier protein is that 50 mg of BSA is dissolved in 6 mL of PBS, and then 3.75 mL of DMF is added, and the dosage is 50 mg.

Preferably, the carrier protein is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

The carbamate hapten has a molecular structure represented by formula (II):

The pyrethroid hapten has a molecular structure represented by formula (III):

Preferably, the carbamate hapten is prepared by the following steps:

S11. Dissolve furan phenol in dichloromethane, then add pyridine to form a mixed solution, and then add p-nitrobenzene chloroformate solution dropwise to the mixed solution (p-nitrobenzene chloroformate is dissolved in dichloromethane), the entire The system is kept in an ice-salt bath below 4°C. After the reaction is completed, the reaction solution is first shaken and washed with dilute hydrochloric acid, and purified by column to obtain the product; the product is a pale yellow crystal;

S12. The product obtained in step S11 is dissolved in tetrahydrofuran to form a tetrahydrofuran mixed solution, 4-aminobutyric acid is dissolved in a sodium bicarbonate solution, and the tetrahydrofuran mixed solution is added dropwise to the sodium bicarbonate solution under an ice bath, and the reaction is completed. Then, continue to add dilute hydrochloric acid under ice bath to adjust the pH of the reaction solution to 4.0, then extract the reaction solution with ethyl acetate, then add anhydrous sodium sulfate to the extract for dehydration, concentrate, and then carry out conventional column purification to collect the target components, and the solvent was distilled off under reduced pressure to obtain the carbamate hapten.

Preferably, the number of times of extracting the reaction solution with ethyl acetate in step S12 is 3 times.

Preferably, the pyrethroid hapten is prepared by the following method:

S21. after carbon tetrachloride, acetonitrile and water are mixed according to the volume ratio of 10:10:15, form the mixture of carbon tetrachloride, acetonitrile and water for subsequent use;

S22. add 2.2 g of cypermethrin, 0.12 g of ruthenium trichloride, 4.3 g of sodium periodate and 35 mL of the mixed solution prepared above in the reaction flask, heat, react, and after the reaction finishes, evaporate the organic solvent to dryness, and the pH of the remaining solution is The value was adjusted to acidic, extracted with ethyl acetate and washed with water; the organic phase was taken and subjected to column purification to obtain the pyrethroid hapten.

Preferably, the heating temperature in step S22 is 60°C, and the reaction time is 2h.

The invention also provides the application of the carbamate and pyrethroid multi-residue antigens in preparing carbamate and pyrethroid multi-residue antibodies or detecting carbamate and pyrethroid pesticide residues.

The present invention also provides a carbamate and pyrethroid multi-residue detection antibody prepared by using the carbamate and pyrethroid multi-residue detection antigen, and the antibody in the detection of carbamate and pyrethroid Applications.

Beneficial effects of the present invention:

The invention fills the long-standing technical gap in the field, and provides a combined antigen for simultaneously detecting the multi-residues of carbamates and pyrethroids. The detection method of ester multi-residue has broad application prospect.

In order to achieve the purpose of the present invention, the present invention provides two haptens at the same time, and provides a preparation method of the hapten, based on the two haptens, the carbamate and chrysanthemum can be prepared very simply and easily. The ester multi-residue specific antigen lays a technical foundation for the detection technical scheme of the present invention.

The preparation method of the invention has mild conditions and is easy to popularize.

Description of drawings

Figure 1. The synthetic route of carbamate haptens.

Figure 2. Mass spectrum of carbamate haptens.

Fig. 3 The synthetic route of pyrethroid hapten.

Figure 4. Mass spectrum of pyrethroid hapten.

Figure 5. Budweiser icELISA standard curve.

Figure 6 Cypermethrin icELISA standard curve.

Detailed ways

The method of the present invention is further described below in conjunction with specific embodiments. The following examples are for illustrative purposes only, and should not be construed as limiting the present invention. Unless otherwise specified, the biological materials and reagent raw materials used in the following examples are conventional commercially available or commercially obtained biological materials and reagent raw materials. Unless otherwise specified, the methods and equipment used in the following examples are routinely used in the field. methods and equipment.

Embodiment 1 The preparation method of carbamates, pyrethroid haptens

(1) Synthesis of carbamate haptens

S11. Weigh 0.8 g of furan phenol and dissolve it in 15 mL of dichloromethane, then add 0.58 mL of pyridine to form a mixed solution, and then add 1 g of p-nitrophenyl chloroformate solution (dissolved in 10 mL of dichloromethane) dropwise into the mixed solution, The reaction was carried out under an ice-salt bath at 4°C for 3 hours; after the reaction, the reaction solution was shaken and washed with 3% hydrochloric acid in a separating funnel, and the washing solution was purified by column to obtain pale yellow crystals.

S12. Take 0.4 g of the above reaction product, dissolve it in 12 mL of tetrahydrofuran, take 0.16 g of 4-aminobutyric acid and dissolve it in 12 mL of sodium bicarbonate solution with pH 8.2, then add the tetrahydrofuran solution dropwise to sodium bicarbonate under ice bath The solution was reacted at room temperature for 4.5h. After the reaction, 4 mol/L hydrochloric acid solution was added under ice bath, the pH of the reaction solution was adjusted to 4, and then the reaction solution was extracted with ethyl acetate for 3 times, and the organic phase was dehydrated with anhydrous Na SO , and then subjected to column purification and reduced pressure. The amino acid methyl ester hapten is obtained by distilling off the solvent. The mass spectrum of the amino acid methyl ester hapten is shown in Figure 2. Prove that it has a molecular structure represented by formula (II):

(2) Synthesis of pyrethroid hapten

S21. After mixing carbon tetrachloride, acetonitrile and water according to the volume of 10:10:15, a mixed solution of carbon tetrachloride, acetonitrile and water is formed;

S22. Take 2.2g of cypermethrin, 0.12g of ruthenium trichloride, 4.3g of sodium periodate and 35mL of the above mixed solution, heat to 60°C for 2h reaction, after the reaction is over, evaporate the organic solvent to dryness, and adjust the pH value of the remaining solution to Acidic, then extracted with ethyl acetate, then washed with water, and the organic phase was taken and purified by column to obtain the pyrethroid hapten. The mass spectrogram of the pyrethroid hapten is shown in Figure 4. Prove that it has the molecular structure of formula (III):

Example 2 Preparation of combined antigens of carbamates and pyrethroids

S1. Weigh 29 mg of the carbamate hapten obtained in Example 1 and dissolve it in 1 mL of DMF, stir and add 36 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC ), 25mg N-N-hydroxysuccinimide (NHS), stirred at room temperature for 8h, after the reaction, 0.25mL of the reaction solution was added to 50mg pH7.4BSA phosphate buffer solution, after stirring at room temperature for 6h, the The reaction solution was put into a dialysis bag, dialyzed with a pH 7.4 phosphate buffer solution for 3 days, and the water was changed 2-3 times a day to obtain the complete carbamate antigen;

S2. Weigh 37 mg of the pyrethroid hapten obtained in Example 1 and dissolve it in 1 mL of DMF, and stir and add 36 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) , 25mg N-N-hydroxysuccinimide (NHS), stirred and reacted at room temperature for 8h to obtain a reaction solution;

S3. Take 0.25 mL of the reaction solution and add it to the DMF solution of carbamate complete antigen. After stirring at room temperature for 6 hours, put it into a dialysis bag and dialyze it with a pH 7.4 phosphate buffer solution for 3 days. Change the water 2 to 3 times a day. , after the dialysis, the multi-residue detection antigens of carbamates and pyrethroids were prepared.

Example 3 Preparation and identification of antibodies

Mix the immunogen with an equal dose of immune adjuvant (Freund's complete adjuvant for the first immunization, and military incomplete Freund's adjuvant for booster immunization), and after complete emulsification with a mixer, the back of the healthy white rabbits was immunized at multiple points. In the future, the immunization was boosted every two weeks, and the serum titer and inhibition rate were determined by indirect ELISA. After the titer was stable, the immunization was boosted again. Cardiac blood was collected 10 days after the last immunization, and the serum was retained by centrifugation. Serum was purified by octanoic acid-ammonium sulfate salting out method to obtain highly specific antibodies.

Example 4 Establishment of ELISA method

The working concentration of antigen and antibody was determined by square array titration. The working concentration of coating was 0.5 μg/mL, and the dilution factor of carbamate and pyrethroid antibody was 1/40000.

Different concentrations of carbamate and pyrethroid solutions were used as experimental solutions, and competition was carried out under gradient dilution, and 9 groups of parallel experiments (n=3) were used. Indirect competitive ELISA method: use the coated ELISA plate with the above working concentration, add the experimental solution and the antibody solution to the small wells of the ELISA plate at the same time, set blank wells and negative control wells at the same time, incubate at 37°C for 40 min, and pour out The liquid in the well was washed 5 times with washing solution, and the ELISA plate was inverted on absorbent paper and tapped: add the diluted ELISA secondary antibody solution, incubate at 37°C for 20 min, wash 5 times with washing solution, and pat dry: add the bottom The color development solution was incubated at 37 °C for 10 min, and the stop solution was added to stop the color development. The absorbance value OD was measured at a wavelength of 450 nm with a microplate reader. The absorbance value OD was used as the ordinate, and the concentration of the carbamate and pyrethroid experimental solution was used. The log10 value of the abscissa is drawn, and the semi-logarithmic standard curve is drawn, as shown in Figure 5 or Figure 6 for the competition standard curve of carbamate and pyrethroid ELISA (respectively, taking carbosulfur and cypermethrin as examples).

The results show that the standard curve has a complete reverse S shape, and has upper and lower platforms. The number of parallel determinations of the standard curve is 3 times. The experiment has good repeatability and the relative standard deviation is within 15%.

The standard curve was drawn by the same method to calculate the lowest detection limit (LOD value), the half inhibition (IC ₅₀ value) and the linear range of other carbamate and pyrethroid pesticides, as shown in Table 1. It shows that the method of the invention has good accuracy and sensitivity, and can be used for the detection of carbamate and pyrethroid in agricultural products and food samples.

Table 1 LOD, IC ₅₀ , LR values of 5 drugs detected by antibodies

drug name LOD(mg/L) IC50(mg/L) LR(mg/L) Budweiser 0.51 61.1 6.6～521.8 Cypermethrin 1.24 74.77 7.88～519.50 Deltamethrin 3.3 143.2 13.2～1556.0 fenothrin 7.54 531.9 15.50～1824.8 Bifenthrin 14.21 824.3 27.19～2499.1

Claims (10)

1. A multi-residue detection antigen of carbamate and pyrethroid has a structural formula shown in formula (I):

the carrier protein is bovine serum albumin.

2. The method for preparing the multi-residue detection antigen of carbamates and pyrethrins as claimed in claim 1, wherein the preparation method comprises the steps of coupling the carbamate hapten with bovine serum albumin by an active ester method, and then continuously coupling the coupled product with the pyrethroid hapten by the active ester method to prepare the multi-residue detection antigen of carbamates and pyrethrins.

3. The method according to claim 2, characterized in that it comprises the following steps:

s1, dissolving carbamate hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction; after the reaction is finished, adding the reaction solution into a phosphate buffer solution of carrier protein with pH of 7.4, continuing the reaction, and dialyzing the reaction solution at room temperature to prepare a complete carbamate antigen;

s2, dissolving the pyrethroid hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction;

s3, dissolving the complete carbamate antigen prepared in the step S1 in a DMF solution, adding the reaction solution prepared in the step S2, reacting at room temperature, and dialyzing the reaction solution after the reaction is finished to obtain the multi-residue detection antigen of carbamates and pyrethrins;

the carbamate hapten has a molecular structure shown in a formula (II):

the pyrethroid hapten has a molecular structure shown in a formula (III):

4. the method according to claim 3, wherein the mass ratio of the carbamate hapten to the carrier protein in the step S1 is 12: 1; the reaction time of the carbamate hapten, EDC and NHS in the step S1 is 8-24 h; and S1, the reaction time of the carbamate hapten and the carrier protein is 6-24 h.

5. The method according to claim 3, wherein the reaction time of step S2 is 8-12 h; the mass ratio of the pyrethroid hapten to the carbamate complete antigen in the step S3 is 10: 1; step S3, the reaction time is 6-8 h; the mass ratio of DMF, EDC and NHS was determined as 10:2: 1.

6. The method of claim 3, wherein the carbamate hapten is prepared by:

s11, dissolving furan phenol in dichloromethane, adding pyridine to form a mixed solution, dropwise adding a p-nitrophenylchloroformate solution dissolved in dichloromethane into the mixed solution, keeping the whole system at a temperature below 4 ℃ in a salt bath, after the reaction is finished, oscillating and washing the reaction solution with dilute hydrochloric acid, and purifying the reaction solution by passing through a column to obtain a product;

s12, dissolving the product obtained in the step S11 in tetrahydrofuran to form a tetrahydrofuran mixed solution, dissolving 4-aminobutyric acid in a sodium bicarbonate solution, dropwise adding the tetrahydrofuran mixed solution into the sodium bicarbonate solution in ice bath, after the reaction is finished, continuously adding dilute hydrochloric acid in the ice bath to adjust the pH of the reaction solution to 4.0, extracting the reaction solution with ethyl acetate, adding anhydrous sodium sulfate into the extracting solution for dehydration, concentrating, performing column purification, collecting a target component, and removing the solvent through reduced pressure distillation to obtain the carbamate hapten.

7. The method of claim 3, wherein the pyrethroid hapten is prepared by:

s21, mixing carbon tetrachloride, acetonitrile and water according to the weight ratio of 10: mixing according to the volume ratio of 10:15 to form a mixture of carbon tetrachloride, acetonitrile and water for later use;

s22, adding 2.2g of cypermethrin, 0.12g of ruthenium trichloride, 4.3g of sodium periodate and 35mL of the prepared mixed solution into a reaction bottle, heating, reacting, evaporating an organic solvent after the reaction is finished, adjusting the pH value of the residual solution to be acidic, extracting with ethyl acetate, and washing with water; purifying the organic phase by a column to obtain the pyrethroid hapten.

8. Use of the carbamate and pyrethroid multi-residue antigen of claim 1 or the carbamate and pyrethroid multi-residue antigen prepared by the method of claims 2 to 7 in the preparation of carbamate and pyrethroid multi-residue antibodies or in the detection of carbamate and pyrethroid pesticide residues.

9. Carbamate and pyrethroid multi-residual antibodies prepared by using carbamate and pyrethroid multi-residual antigens according to claim 1 or carbamate and pyrethroid multi-residual antigens prepared by the method according to claims 2 to 7.

10. Use of the multi-residue antibodies of carbamates and pyrethrins according to claim 9 for the detection of carbamates and pyrethrins.

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